Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre-purification by foam separation during cultivation

Kirsten Kottmeier*, Tobias Jan Günther, Jost Weber, Susann Kurtz, Kai Ostermann, Gerhard Rödel, Thomas Bley

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Hydrophobins are small surface-active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor-made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha-factor prepro-peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre-purification using foam separation.

Original languageEnglish
JournalEngineering in Life Sciences
Volume12
Issue number2
Pages (from-to)162-170
Number of pages9
ISSN1618-0240
DOIs
Publication statusPublished - 2012
Externally publishedYes

Keywords

  • Foam separation
  • GAP promoter
  • HFB1
  • Hydrophobin
  • Pichia pastoris

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